Universal enzyme responsive linker for assembling ligands on DNA functionalized nanomaterials

ABSTRACT

Described herein is an enzyme-mediated approach to bioconjugation at nanoparticle (NP) surfaces. This process is enabled by a new synthetic linker compatible with the covalent attachment of alkyne modified substrates, including dyes, peptides and nucleic acids. The methods described herein specifically allow for the linkage of molecules to a DNA-functionalized nanoparticle surface. Enzymatic ligation of molecules to the terminal hydroxyl group of DNA using T4 DNA ligase is achieved through incorporation of a single monophosphate on the approaching substrate. In contrast to previous strategies, the linkers disclosed herein are compatible with alkyne modified molecules of a variety of sizes and charges indicating that the ligase minimally requires the monophosphate and the incoming hydroxyl for conjugation to be successful.

CROSS-REFERENCE TO RELATED-APPLICATIONS

The present application is a divisional application of U.S. patentapplication Ser. No. 15/917,911 filed Mar. 12, 2018, which claimspriority to U.S. Provisional Patent Application No. 62/470,327 entitled“UNIVERSAL ENZYME RESPONSIVE LINKER FOR ASSEMBLING LIGANDS ON DNAFUNCTIONALIZED NANOMATERIALS” filed Mar. 12, 2017, each of which isincorporated by reference in its entirety.

SEQUENCE LISTING

The sequence listing submitted herewith, entitled:

“16-1821-US-DIV_SequenceListing_ST25.txt” and 2 kb in size, isincorporated by reference in its entirety.

BACKGROUND

Surface functionalization of inorganic nanoparticles with DNA, RNA,peptides and dyes is needed for the assembly of nanoscale materials fora variety of applications ranging from biosensing, to bioimaging, andtherapeutics. A critical step in building these hybrid biomaterials isthe ability to present diverse chemical functionalities at ananoparticles surface efficiently and in high enough yields to assemblediverse ligands. Chemical bioconjugation strategies, including the morecommon EDC approach for amide coupling and the use of thiol reactivelinkers have been predominantly used for attaching peptides, dyes andnucleic acids to nanoparticles (NPs). This is attributed to theircompatibility with aqueous conditions and their tolerance for highersalt concentrations that biomolecules often require to maintain theirfolded structures. However, such chemical conjugation strategies oftenrequire the pairing of a different chemical modification for eachindividual substrate to be attached to a NPs surface depending on thereactive sites available for functionalization.

In light of this multistep synthetic approach to nanoparticlefunctionalization, a generalized chemical approach was developed that isboth biologically compatible and straightforward. A DNA functionalizedNP (DNA-NP) was used as a platform on which to test the enzyme-mediatedapproach to nanoparticle functionalization. DNA-NPs have gainedsignificant attention in the last two decades, owning to their ease ofsynthesis and chemical stability in aqueous environments.Hybridization-based assembly of nanomaterials takes advantage of theWatson-crick base pairing interactions of DNA's double helicalstructure.

To date there is no general enzymatic strategy for modification of theDNA on a DNA-nanoparticle to impart compatibility for chemical ligationbetween the DNA and a molecule of non-nucleic acid structure. Themethods and compositions disclosed herein use the terminal ends of DNAmolecules, in particular, the 3′OH end recognized by ligase enzymes.Disclosed herein are compositions that can interface chemicalmodifications with the specificity of the ligase. This versatileapproach is important for the rapidly growing field of nucleic acidbased therapeutics and offers an important strategy aimed at repurposingenzymes for use as assembly tools for functionalizing nanomaterials in achemically specific manner.

SUMMARY

Disclosed herein are compositions and methods that enablefunctionalizing molecules with enzymes in an efficient manner (incontrast to multi-step synthetic chemical modification of biomolecules).Using the compositions and methods as disclosed herein, enzymes, withhigh specificity, can rapidly and covalently attach any alkyne,dibenzocyclooctyne group (DBCO) or other compatible “click” moleculeonto DNA using T4 DNA ligase.

In one aspect, the disclosure is related to a heterobifunctional linkerhaving the general formula: (HO)2(O)P—O—I—Y; wherein I is an interveningmoiety, the intervening moiety having a molecular weight in the range of100 to 10000; and Y is a chemically-reactive moiety, thechemically-reactive moiety being reactable to couple the linker to anorganic compound in aqueous solution, or a salt thereof, thechemically-reactive moiety and intervening moieties being selected suchthat the heterobifunctional linker or salt thereof is water soluble at aconcentration of at least 10 pM at a pH within the range of 6.5 to 7.8.In some embodiments, the chemically-reactive moiety is reactable tocouple the linker to a biomolecule in aqueous solution.

In a second aspect, the disclosure relates to a composition comprisingthe heterobifunctional linker as disclosed herein, wherein the phosphatemoiety of the heterobifunctional linker is covalently bound to a firstmolecule. In some embodiments, the first molecule is a nucleic acid, andthe phosphate moiety of the heterobifunctional linker is covalentlybound to a 3′-hydroxyl of a phosphate moiety of the nucleic acid to forma phosphodiester. In certain embodiments, the chemically-reactive moietyof the heterobifunctional linker is covalently bound to a secondmolecule.

In a third aspect, the disclosure relates to a composition comprisingthe heterobifunctional linker as disclosed herein, wherein thechemically-reactive moiety of the heterobifunctional linker iscovalently bound to a second molecule.

In a fourth aspect, the disclosure relates to a method of covalentlylinking two molecules comprising:

(a) reacting the chemically-reactive moiety of the heterobifunctionallinker as disclosed herein with a first molecule comprising a functionalgroup capable of covalently binding the chemically-reactive moiety ofthe heterobifunctional linker, wherein the reacting occurs underconditions and for a time suitable to covalently bind the first compoundto the chemically-reactive moiety of the heterobifunctional linker; and

(b) reacting the phosphate moiety of the heterobifunctional linker witha second molecule comprising a 3′-OH group of a nucleic acid phosphatein the presence of a T4 DNA ligase for a time and under conditions, toligate the second molecule to the phosphate moiety of the first complexto form a phosphodiester bond between the nucleic acid phosphate and thephosphate moiety of the heterobifunctional linker.

In a fifth aspect, the disclosure relates to a kit comprising:

-   -   (a) the heterobifunctional linker as disclosed herein;    -   (b) reagents for reacting a first molecule to the reactive        moiety of the heterobifunctional linker; and    -   (c) a T4 DNA ligase and reagents to ligate a second molecule to        the phosphate moiety of the heterobifunctional linker.

The present disclosure provides the following non-limiting numberedembodiments:

Embodiment 1

A heterobifunctional linker having the general formula:(HO)2(O)P—O—I—Y;wherein

-   -   I is an intervening moiety, the intervening moiety having a        molecular weight in the range of 100 to 10000; and    -   Y is a chemically-reactive moiety, the chemically-reactive        moiety being reactable to couple the linker to an organic        compound in aqueous solution,        or a salt thereof, the chemically-reactive moiety and        intervening moieties being selected such that the        heterobifunctional linker or salt thereof is water soluble at a        concentration of at least 10 pM at a pH within the range of 6.5        to 7.8.

Embodiment 2

The heterobifunctional linker according to embodiment 1, wherein thechemically-reactive moiety is reactable to couple the linker to abiomolecule in aqueous solution.

Embodiment 3

The heterobifunctional linker according to embodiment 1 or embodiment 2,wherein the chemically-reactive moiety comprises an azide.

Embodiment 4

The heterobifunctional linker according to embodiment 1 or embodiment 2,wherein the chemically-reactive moiety comprises an alkyne.

Embodiment 5

The heterobifunctional linker according to embodiment 4, wherein thealkyne has the structural formula:

Embodiment 6

The heterobifunctional linker according to embodiment 4, wherein thealkyne has the structural formula:

Embodiment 7

The heterobifunctional linker according to embodiment 1 or embodiment 2,wherein the chemically-reactive moiety comprises a maleimide.

Embodiment 8

The heterobifunctional linker according to embodiment 1 or embodiment 2,wherein the chemically-reactive moiety comprises a cyclopentadiene.

Embodiment 9

The heterobifunctional linker according to embodiment 1 or embodiment 2,wherein the chemically-reactive moiety comprises a thiol.

Embodiment 10

The heterobifunctional linker according to embodiment 1 or embodiment 2,wherein the chemically-reactive moiety comprises an alkene.

Embodiment 11

The heterobifunctional linker according to embodiment 10, wherein thechemically-reactive moiety is a terminal alkene.

Embodiment 12

The heterobifunctional linker according to embodiment 1 or embodiment 2,wherein the chemically-reactive moiety comprises a carboxylate.

Embodiment 13

The heterobifunctional linker according to embodiment 1 or embodiment 2,wherein the chemically-reactive moiety comprises an amine.

Embodiment 14

The heterobifunctional linker according to embodiment 1 or embodiment 2,wherein the chemically-reactive moiety comprises a carboxylate ester ofN-hydroxysuccinimide.

Embodiment 15

The heterobifunctional linker according to embodiment 1 or embodiment 2,wherein the chemically-reactive moiety comprises an isocyanate or anisothiocyanate.

Embodiment 16

The heterobifunctional linker according to embodiment 1 or embodiment 2,wherein the chemically-reactive moiety comprises a maleimide, aniodoacetamide, a (pyridin-2-yl)disulfanyl, or a(3-carboxy-4-nitrophenyl)disulfanyl.

Embodiment 17

The heterobifunctional linker according to embodiment 1, wherein thechemically-reactive moiety is reactive in a cycloaddition, e.g., a 3+2cycloaddition or a 4+2 cycloaddition, at a temperature below 60° C.

Embodiment 18

The heterobifunctional linker according to any of embodiments 1-17,wherein the intervening moiety comprises, consists essentially of, or isa polyethylene glycol.

Embodiment 19

The heterobifunctional linker according to embodiment 18, wherein theintervening moiety comprises, consists essentially of, or is apolyethyelene glycol having structure —(CH₂CH₂O)_(n)— in which n is inthe range of 3-200, e.g., 3-150, or 3-100, or 3-50, or 3-20, or 5-200,or 5-150, or 5-100, or 5-50, or 10-200, or 10-150, or 10-100.

Embodiment 20

The heterobifunctional linker according to embodiment 19, wherein theintervening moiety comprises the structure —(CH₂CH₂O)_(n)—(CH₂)_(m)— inwhich m is in the range of 1-6, e.g., 2-6, or 1-4, or 2-4, or is 2 or 3.

Embodiment 21

The heterobifunctional linker according to any of embodiments 1-17,wherein the intervening moiety comprises, consists essentially of, or isa polyethylene imine.

Embodiment 22

The heterobifunctional linker according to embodiment 21, wherein thepolyethyleneimine has a molecular weight in the range of 100-5000, e.g.,100-2000, or 100-1000, or 100-500, or 200-5000, or 200-2000, or200-1000.

Embodiment 23

The heterobifunctional linker according to any of embodiments 1-17,wherein the intervening moiety comprises, consists essentially of, or isa copolymer or cooligomer of ethylene glycol and ethyleneimine, forexample, having a molecular weight in the range of 100-5000, e.g.,100-2000, or 100-1000, or 100-500, or 200-5000, or 200-2000, or200-1000.

Embodiment 24

The heterobifunctional linker according to any of embodiments 1-17,wherein the intervening moiety comprises, consists essentially of, or isa peptide oligomer or polypeptide, for example, having a molecularweight in the range of 100-5000, e.g., 100-2000, or 100-1000, or100-500, or 200-5000, or 200-2000, or 200-1000.

Embodiment 25

The heterobifunctional linker according to any of embodiments 1-17,wherein the intervening moiety comprises, consists essentially of, or isa polyester of one or more of lactic acid and glycolic acid, forexample, having a molecular weight in the range of 100-5000, e.g.,100-2000, or 100-1000, or 100-500, or 200-5000, or 200-2000, or200-1000.

Embodiment 26

The heterobifunctional linker according to embodiment 25, wherein theintervening moiety comprises a polyester of one or more of lactic acidand glycolic acid, and wherein the polyester comprises poly(lacticacid-co-glycolic acid).

Embodiment 27

The heterobifunctional linker according to any of embodiments 1-17,wherein the intervening moiety comprises, consists essentially of, or isa poly(propylene glycol), for example, having a molecular weight in therange of 100-2000, 100-1000, or 100-500.

Embodiment 28

The heterobifunctional linker according to any of embodiments 1-17,wherein the intervening moiety comprises, consists essentially of, or isa poly(ethylene glycol-co-propylene glycol), for example, having amolecular weight in the range of 100-5000, e.g., 100-2000, or 100-1000,or 100-500, or 200-5000, or 200-2000, or 200-1000.

Embodiment 29

The heterobifunctional linker according to any of embodiments 1-28,wherein the intervening moiety comprises the structure —(L)—(CH₂)_(m)—in which m is in the range of 1-6, e.g., 2-6, or 1-4, or 2-4, or is 2 or3 and in which L is a polyethylene glycol, a polyethylene imine, acopolymer or cooligomer of polyethylene glycol and polyethyleneimine, apeptide oligomer or polypeptide, a polyester of one or more of lacticacid and glycolic acid, a poly(propylene glycol), or a poly(ethyleneglycol-co-propylene glycol).

Embodiment 30

The heterobifunctional linker according to any of embodiments 1-29,wherein there are no branched carbon atoms or nitrogen atoms within 4atoms of, or within 6 atoms of, or within 8 atoms of, or within 10 atomsof the phosphate, a branched carbon atom or nitrogen atom being definedas having more than three non-hydrogen, non-carbonyl substituentsdirectly bound thereto.

Embodiment 31

The heterobifunctional linker according to any of embodiments 1-30,wherein the heterobifunctional linker has a molecular weight in therange of 100-7000, or 100-5000, or 100-2000, or 100-1000, or 100-500, or200-10000, or 200-7000, or 200-5000, or 200-2000, or 200-1000, or200-500, or 500-10000, or 500-7000, or 500-5000, or 500-2000, or1000-10000, or 1000-7000, or 1000-5000.

Embodiment 32

The heterobifunctional linker according to embodiment 1, having thestructure:

Embodiment 33

A composition comprising the heterobifunctional linker of any one ofembodiments 1-32, wherein the phosphate moiety of the heterobifunctionallinker is covalently bound to a first molecule.

Embodiment 34

The composition according to embodiment 33, wherein the first moleculecomprises a nucleic acid, and wherein the phosphate moiety of theheterobifunctional linker is covalently bound to a 3′-hydroxyl of aphosphate moiety of the nucleic acid to form a phosphodiester.

Embodiment 35

The composition according to embodiment 34, wherein the nucleic acid isa DNA of a DNA-coated nanoparticle.

Embodiment 36

A composition according to any of embodiments 33-35, wherein thereactive moiety of the heterobifunctional linker is covalently bound toa second molecule.

Embodiment 37

A composition comprising the heterobifunctional linker of any one ofembodiments 1-32, wherein the chemically-reactive moiety of theheterobifunctional linker is covalently bound to a second molecule.

Embodiment 38

A composition according to embodiment 36 or embodiment 37, wherein thesecond molecule is, comprises, or consists essentially of a polypeptide,such as a protein.

Embodiment 39

A composition according to embodiment 36 or embodiment 37, wherein thesecond molecule is, comprises or consists essentially of a nucleic acid,such as a DNA or an RNA.

Embodiment 40

A composition according to embodiment 36 or embodiment 37, wherein thesecond molecule is, comprises, or consists essentially of a dye.

Embodiment 41

A composition according to embodiment 36 or embodiment 37, wherein thesecond molecule comprise a lipid, a sterol, fatty acids, or a polymer.

Embodiment 42

A composition according to any of embodiments 36-41, wherein thechemically-reactive moiety of the heterobifunctional linker comprises anazide, and the heterobifunctional linker is covalently bound to thesecond molecule through a thiazole formed from the azide and an alkyne.

Embodiment 43

A composition according to any of embodiments 36-41, wherein thechemically-reactive moiety of the heterobifunctional linker comprises analkyne, and the heterobifunctional linker is covalently bound to thesecond molecule through a thiazole formed from the alkyne and an azide.

Embodiment 44

A composition according to any of embodiments 36-41, wherein thechemically-reactive moiety of the heterobifunctional linker comprise acyclopentadiene, and the heterobifunctional linker is covalently boundto the second molecule through a cycloaddition product of thecyclopentadiene.

Embodiment 45

A composition according to any of embodiments 36-41, wherein thechemically-reactive moiety of the heterobifunctional linker comprise athiol, and the heterobifunctional linker is covalently bound to thesecond molecule through a thiol-ene or thiol-yne reaction product of thethiol.

Embodiment 46

A composition according to any of embodiments 36-41, wherein thechemically-reactive moiety of the heterobifunctional linker comprises analkene, e.g., a terminal alkene, and the heterobifunctional linker iscovalently bound to the second molecule through a thiol-ene reactionproduct of the alkene.

Embodiment 47

A composition according to any of embodiments 36-41, wherein thechemically-reactive moiety of the heterobifunctional linker comprise amaleimide, and the heterobifunctional linker is covalently bound to thesecond molecule through a thiol-ene reaction product of the maleimide.

Embodiment 48

A composition according to any of embodiments 36-41, wherein thechemically-reactive moiety of the heterobifunctional linker comprises analkyne, e.g., a terminal alkyne, and the heterobifunctional linker iscovalently bound to the second molecule through a thiol-ene reactionproduct of the alkyne.

Embodiment 49

A composition according to any of embodiments 36-41, wherein thechemically-reactive moiety of the heterobifunctional linker comprise acarboxylate and the heterobifunctional linker is covalently bound to thesecond molecule through a condensation reaction product of thecarboxylate (e.g., with an amine to form an amide).

Embodiment 50

A composition according to any of embodiments 36-41, wherein thechemically-reactive moiety of the heterobifunctional linker comprises anand the heterobifunctional linker is covalently bound to the secondmolecule through a condensation reaction product of the amine (e.g.,with a carboxylate to form an amide).

Embodiment 51

A composition according to any of embodiments 36-41, wherein thechemically-reactive moiety of the heterobifunctional linker comprise acarboxylate ester of N-hydroxysuccinimide and the heterobifunctionallinker is covalently bound to the second molecule through a condensationreaction product of the a carboxylate ester of N-hydroxysuccinimide(e.g., with an amine to form an amide).

Embodiment 52

A composition according to any of embodiments 36-41, wherein thechemically-reactive moiety of the heterobifunctional linker comprises anisocyanate or an isothiocyanate and the heterobifunctional linker iscovalently bound to the second molecule through a reaction product ofthe isocyanate or isothiocyanate (e.g., with an amine such as a lysineamine to form a urea or a thiourea).

Embodiment 53

A composition according to any of embodiments 36-41, wherein thechemically-reactive moiety of the heterobifunctional linker comprises aniodoacetamide and the heterobifunctional linker is covalently bound tothe second molecule through a reaction product of the iodoacetamide(e.g., with a thiol such as a cysteine thiol to form a thioether).

Embodiment 54

A composition according to any of embodiments 36-41, wherein thechemically-reactive moiety of the heterobifunctional linker comprise a(pyridin-2-yl)disulfanyl or a (3-carboxy-4-nitrophenyl)disulfanyl andthe heterobifunctional linker is covalently bound to the second moleculethrough a reaction product of the (pyridin-2-yl)disulfanyl or(3-carboxy-4-nitrophenyl)disulfanyl iodoacetamide (e.g., with a thiolsuch as a cysteine thiol to form a disulfide bridge).

Embodiment 55

A composition according to any of embodiments 36-41, wherein thechemically-reactive moiety of the heterobifunctional linker is reactivein a cycloaddition (e.g., a [3+2] cycloaddition or a [4+2]cycloaddition) at a temperature below 60° C., and the heterobifunctionallinker is covalently bound to the second molecule through acycloaddition reaction product of the chemically-reactive moiety.

Embodiment 56

A method of covalently linking two molecules (e.g., to form acomposition according to any of embodiments 36 and 38-55) comprising:

-   -   (a) reacting the chemically-reactive moiety of the        heterobifunctional linker of any of claims 1-21 with a first        molecule comprising a functional group capable of covalently        binding the chemically-reactive moiety of the heterobifunctional        linker, wherein the reacting occurs under conditions and for a        time suitable to covalently bind the first compound to the        chemically-reactive moiety of the heterobifunctional linker; and    -   (b) reacting the phosphate moiety of the heterobifunctional        linker with a second molecule comprising a 3′-OH group of a        nucleic acid phosphate in the presence of a T4 DNA ligase for a        time and under conditions, to ligate the second molecule to the        phosphate moiety of the first complex to form a phosphodiester        bond between the nucleic acid phosphate and the phosphate moiety        of the heterobifunctional linker.

Embodiment 57

The method of embodiment 56, wherein step (a) is performed before step(b).

Embodiment 58

The method of embodiment 57, wherein the product of step (a) is purified(e.g., using chromatography) before step (b) is performed.

Embodiment 59

The method of any of embodiments 56-58, wherein step (a) is performedusing copper catalysis.

Embodiment 60

A kit comprising:

-   -   (a) the heterobifunctional linker of any of embodiments 1-32;    -   (b) reagents for reacting a first molecule to the reactive        moiety of the heterobifunctional linker; and    -   (c) a T4 DNA ligase and reagents to ligate a second molecule to        the phosphate moiety of the heterobifunctional linker.

These and other features and advantages of the present disclosure willbe more fully understood from the following detailed description of theinvention taken together with the accompanying claims. It is noted thatthe scope of the claims is defined by the recitations therein and not bythe specific discussion of features and advantages set forth in thepresent description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following figures are in accordance with example embodiments.

FIG. 1 shows a schematic representation of the heterobifunctional linkerthat presents an azide for copper catalyzed click chemistry and amonophosphate for enzymatic ligation.

FIG. 2 shows an exemplary four step synthesis from commerciallyavailable reagents that results in a monophosphorylated linker and anazide group that can be used for copper catalyzed click reactions.

FIG. 3 shows alkyne modification of peptides. Alkyne modification allowsfor covalent attachment of a peptide to the monophosphorylated linker.Once purified, the alkyne modified peptide can be attached to the azideof the monophosphorylated linker forming the tetrazole linkage.

FIG. 4A shows an exemplary size and charge analysis of the productsgenerated through covalent enzymatic attachment. An agarose gel shiftassay demonstrates the successful linkage of a short peptide (CLPKTGGR;SEQ ID NO:01) to the surface of a gold nanoparticle.

FIG. 4B shows an exemplary size and charge analysis of the productsgenerated through covalent enzymatic attachment. A dynamic lightscattering analysis of the peptide conjugated gold nanoparticledemonstrates an increase in overall size of the particles.

FIG. 5 shows a gel shift assay showing the change in particle size postenzymatic ligation of linker modified peptide, DNA, and dye. Lane 1DNA-gold NPs; Lane 2 DNA-NPs post ligation to DNA strand; Lane 3 postligation to peptide; and Lane 4 post ligation to Cy3 fluorophore usingthe universal linker. Ligation reaction conditions: 24 hours, 37° C.,Gel conditions: 1% Agarose, 10 minutes at 200V.

FIG. 6 shows confocal microscopy of HeLa cells incubated with dyelabeled DNA-gold nanoparticles (NP) functionalized using the universalmonophosphorylated linker. Cy3-alkyne linked to the universal linker wasligated to the surface of a DNA functionalized NP and incubated in HeLacells (2 nm NPs).

FIG. 7 shows an exemplary schematic for a universal linker enablingenzyme-mediated attachment of nucleic acids to nanoparticle surfaces(i.e., gold). Anchor DNA 5′-TTT TTT TTT TCA ATC ACA ACC A-3′ (SEQ IDNO:02); Bridge 5′-CGT CAT CAA CGA TGG TTG TGA TTG-3′ (SEQ ID NO:03);Ligation DNA: 5′-TCG TTG ATG ACG CTA GCT AGA C-3′ (SEQ ID NO:04);mp-Ligation DNA: 5′-TCG TTG ATG ACG CTA GCT AGA C-3′ (SEQ ID NO:08)where mp=monophosphorylated as the nucleic acid can bemonophosphorylated via attachment of the universal linker.

FIG. 8 shows a schematic for the use of the universal linker enablingenzyme-mediated attachment of ligands to nanoparticle surfaces (i.e.,gold). Alkylo-CLPKTGGR (SEQ ID NO:05); mp-CLPKTGGR (SEQ ID NO:07) wheremp=monophosphorylated as the peptide can be monophosphorylated viaattachment of the universal linker.

FIG. 9A shows the synthesis of alkylo-CLPKTGGR (SEQ ID NO:05) fromCLPKTGGR (SEQ ID NO:01). Propargylmaleimide (2.4 mg, 0.018 mmol) wasadded to a solution of sortase recognition motif (CLPKTGGR; SEQ IDNO:01) (10 mg, 0.012 mmol) in 200 μL of 5% acetonitrile in 50 mM HEPESbuffer (pH=7.2), and the solution stirred at room temperature for 2hours.

FIG. 9B shows the ¹H NMR 300 of compound alkylo-CLPKTGGR (SEQ ID NO:05)in d-methanol/CDCl₃.

FIG. 10A shows the synthesis of compound 1. Tosyl chloride (1.00 g, 5.25mmol) was added at 5° C. to a solution of TEG (triethylene glycol, 1.57g, 10.5 mmol) in anhydrous methylene chloride (6 mL). This was followedby drop-wise addition of DIPEA (N,N-diisopropylethylamine, 1.0 mL, 5.77mmol). The water bath was removed and the reaction mixture stirred atroom temperature for 18 hours. The resulting mixture was diluted withwater (6 mL) and washed with 1 M HCl, brine (2×10 mL), and water (3×10mL). The solution was dried over sodium sulfate and concentrated undervacuum. The resulting residue was purified by silica gel chromatography(ethyl acetate/hexane 7:3) to obtain the product as yellow oil (1.26 g,79%). LEN et al., “Micellar Catalysis Using a Photochromic Surfactant:Application to the Pd-Catalyzed Tsuji-Trost Reaction in Water” J. Org.Chem., 79(2): 493-500 (2014).

FIG. 10B shows the ¹H NMR 300 of compound 1 CDCl₃.

FIG. 11 shows the synthesis of compound 2 (Jka-01-003). Sodium azide(2.69 g, 41.4 mmol) was added to a solution of compound 1 (1.26 g, 4.14mmol) in anhydrous dimethylformamide (DMF, 15 mL), and the mixturestirred at 60° C. for 16 hours. The mixture was diluted with water (20mL) and the organic layer extracted with ethyl acetate (3×15 mL). Theorganic layer was washed with brine (10 mL), followed by water (2×10mL), and dried under sodium sulfate to yield the product as brown oil.

FIG. 12A shows the synthesis of compound 3. Triethylamine (0.3 mL, 2.13mmol) was added dropwise under argon atmosphere to a 5 mLtetrahydrofuran (THF) solution of phosphoryl chloride (POCl₃, 180 μL,1.94 mmol) in 50 mL round bottomed flask at 0° C. The resultant mixturewas stirred for 20 minutes until a white solid precipitated. On additionof compound 2 (0.26 g, 1.48 mmol) dissolved in 1 mL anhydrous THF, theprecipitate dissolved. After 2 hours, the THF was removed under reducedpressure and the sample purified by preparative TLC usingmethanol/methylene chloride (1:20) as eluent to afford the titlecompound as colorless oil. LU et al., “Carboxyl-polyethyleneglycol-phosphoric acid: a ligand for highly stabilized iron oxidenanoparticles” J. Mater. Chem., 22:19806-19811 (2012). GNAUCK et al.,“Carboxy-Terminated Oligo(ethylene glycol)-Alkane Phosphate: Synthesisand Self-Assembly on Titanium Oxide Surfaces” Langmuir 23(2):377-381(2007).

FIG. 12B shows an ESI-MS of compound 3. ESI-HRMS (m/z): EM-Hfcalculation for C₆H₁₃N₃O₆P⁻ 254.0547; found, 254.0578.

DETAILED DESCRIPTION

The heterobifunctional linkers described here allow the ability toenzymatically attach, for example, a nucleic acid molecule to a diverserange of molecules or nanomaterials (these include but are not limitedto: peptides, enzymes, antibodies, dyes, nucleic acids) with variouschemical modifications through a single heterobiofunctional linker.These linkers, for example, present an azide on one end for utilizingthe copper catalyzed alkyne-azide cycloaddition (CuAAC) “click”chemistry approach and at the other end a terminal phosphate moiety forligation to the 3′ hydroxyl of a DNA molecule by the T4 DNA ligaseenzyme. This is advantageous as it opens the door to a variety ofchemical modifications through a single robust enzyme approach.

For example, the heterobifunctional linkers disclosed herein can be usedfor the selective functionalization of DNA-coated nanoparticles (NPs).DNA functionalized materials are an extremely attractive platform fordrug delivery applications, biosensing and bioimaging. Using DNA whichpresents a terminal 3′ hydroxyl DNA can be functionalized using T4 DNAligase, ATP, and the monophosphorylated heterobiofunctional linkerdescribed herein to obtain the covalent attachment of the molecule ofinterest to DNA under mild, biocompatible conditions. This linker canaccelerate the surface modification of substrate-based technologies aswell (e.g., the selective, efficient assembly of biomolecules on achemically modified surface). For example, an alkyne modified peptidecan be “clicked” to the heterobifunctional linker disclosed herein andthen enzymatically attached to DNA linkers affixed to either ananoparticle surface or a substrate using T4 DNA ligase.

The DNA functionalized nanomaterial is used as an example substrate, butcould be replaced by any kind of chip assay or substrate to which DNA isattached at its 5′ end to the surface. The results show that the minimalrequirements for the success of the linker to attach an R ground (whereR can be DNA, peptide or dye/fluorophore) using the linker to a DNAmolecule is the presence of the monophosphate group bound to a flexibleintervening moiety (for example, triethylene glycol).

In one aspect, the disclosure is related to a heterobifunctional linkerhaving the general formula: (HO)2(O)P—O—I—Y; wherein

-   -   I is an intervening moiety, the intervening moiety having a        molecular weight in the range of 100 to 10000; and    -   Y is a chemically-reactive moiety, the chemically-reactive        moiety being reactable to couple the linker to an organic        compound in aqueous solution, or a salt thereof, the        chemically-reactive moiety and intervening moiety being selected        such that the heterobifunctional linker or salt thereof is water        soluble at a concentration of at least 10 pM at a pH within the        range of 6.5 to 7.8.

As used herein, the term “chemically-reactive moiety” refers to anymoiety that is reactable to covalently couple the linker to an organiccompound. For covalent attachment, the chemistry should be compatiblewith terminal amines or carboxylic acids when considering the attachmentof a peptide to the hydroxyl or phosphate groups of nucleic acidmolecule. As the reactive chemistry for DNA is limited, typically DNA isfirst modified with a chemical leaving group that would make itsattachment to the peptide suitable with chemistries such asethyl(dimethylaminopropyl) carbodiimide, N-hydroxysuccinimide (EDC-NHS)coupling chemistry or thiol reactive chemistry.

The chemically reactive moiety can be, for example, a moiety that canparticipate in a so-called “click” reaction. Click reactions are knownto be reactions that are high yielding, wide in scope, create onlybyproducts that can be removed without chromatography, are simple toperform, and can be conducted in easily removable or benign solvents.While a variety of such chemistries are described below, the person ofordinary skill in the art will appreciate that other chemistries can beadapted for use in the systems described herein.

In some embodiments, the chemically-reactive moiety is reactable tocouple the linker to a biomolecule in aqueous solution. In certainembodiments, the chemically-reactive moiety comprises an azide. In anembodiment, the chemically-reactive moiety comprises an alkyne (e.g., astrained alkyne such as an alkyne in a ring), for example, having hasthe structural formula:

In another embodiment, the alkyne has the structural formula:

In some embodiments of the heterobifunctional linker, thechemically-reactive moiety comprises a molecule selected from the groupconsisting of an alkene, an amine, a maleimide, a carboxylate, acarboxylate ester of N-hydroxysuccinimide, a(3-carboxy-4-nitrophenyl)disulfanyl, a cyclopentadiene, aniodoacetamide, an isocyanate, isothiocyanate, a(pyridin-2-yl)disulfanyl, and a thiol. In an embodiment, thechemically-reactive moiety comprises an alkene and the alkene is aterminal alkene.

In some embodiments of the heterobifunctional linker, thechemically-reactive moiety is reactive in a cycloaddition at atemperature below 60° C. In non-limiting examples, the cycloaddition isa [3+2] cycloaddition or a [4+2] cycloaddition.

As used herein, the term “intervening moiety” refers to any structure ormolecule that joins phosphate moiety of heterobifunctional linker to thechemically-reactive moiety. Such an intervening moiety or moieties canalso be referred to as a bridge, or a spacer, moiety or moieties. Insome embodiments, the intervening moiety comprises an entity selectedfrom the group consisting of a polyethylene glycol, a polyethyleneimine,a copolymer or cooligomer of ethylene glycol and ethyleneimine, apeptide oligomer or polypeptide, a polyester of one or more of lacticacid and glycolic acid, a poly(propylene glycol), and a poly(ethyleneglycol-co-propylene glycol).

In certain embodiments, the intervening moiety comprises a polyethyeleneglycol having structure —(CH₂CH₂O)_(n)— in which n is in the range of3-200. In some embodiments, n is in the range of 3-150, or 3-100, or3-50, or 3-20, or 5-200, or 5-150, or 5-100, or 5-50, or 10-200, or10-150, or 10-100. In an embodiment, the intervening moiety comprisesthe structure —(CH₂CH₂O)_(n)—(CH₂)_(m)— in which m is in the range of1-6 (for example, m is in the range of 2-6, or 1-4, or 2-4, or m is 2 or3).

In certain embodiments, the intervening moiety comprises, consistsessentially of, or is a polyethylene imine, and the polyethyleneiminecan have a molecular weight in the range of 100-5000, for example,100-2000, or 100-1000, or 100-500, or 200-5000, or 200-2000, or200-1000.

In some embodiments, the intervening moiety comprises, consistsessentially of, or is a copolymer or cooligomer of ethylene glycol andethyleneimine, for example, having a molecular weight in the range of100-5000, for example, 100-2000, or 100-1000, or 100-500, or 200-5000,or 200-2000, or 200-1000.

In certain embodiments, the intervening moiety comprises, consistsessentially of, or is a peptide oligomer or polypeptide, for example,having a molecular weight in the range of 100-5000, for example,100-2000, or 100-1000, or 100-500, or 200-5000, or 200-2000, or200-1000. The peptide oligomer or polypeptide can be formed from, forexample, natural amino acids, unnatural amino acids, or a combinationthereof. In certain embodiments, the peptide oligomer or polypeptide isformed from natural L-amino acids. In other embodiments, the peptideoligomer or polypeptide is formed from D-amino acids (e.g., enantiomersof natural L-amino acids) or from a combination of D- and L-amino acids.

In some embodiments, the intervening moiety comprises, consistsessentially of, or is a polyester of one or more of lactic acid andglycolic acid, for example, having a molecular weight in the range of100-5000, for example, 100-2000, or 100-1000, or 100-500, or 200-5000,or 200-2000, or 200-1000. In some embodiments, the polyester ispoly(lactic acid-co-glycolic acid).

In certain embodiments, the intervening moiety comprises, consistsessentially of, or is a poly(propylene glycol), for example, having amolecular weight in the range of 100-2000, 100-1000, or 100-500.

In some embodiments, the intervening moiety comprises, consistsessentially of, or is a poly(ethylene glycol-co-propylene glycol), forexample, having a molecular weight in the range of 100-5000, forexample, 100-2000, or 100-1000, or 100-500, or 200-5000, or 200-2000, or200-1000.

In an embodiment, the intervening moiety comprises the structure—(L)—(CH₂)_(m)— in which m is in the range of 1-6 (e.g., 2-6, or 1-4, or2-4, or is 2 or 3), and in which L comprises a polyethylene glycol, apolyethyleneimine, a copolymer or cooligomer of polyethylene glycol andpolyethyleneimine, a peptide oligomer or polypeptide, a polyester of oneor more of lactic acid and glycolic acid, a poly(propylene glycol), or apoly(ethylene glycol-co-propylene glycol).

In some embodiments of the heterobifunctional linker as disclosedherein, there are no branched carbon atoms or nitrogen atoms within 4atoms of, or within 6 atoms of, or within 8 atoms of, or within 10 atomsof the phosphate, a branched carbon atom or nitrogen atom being definedas having more than three non-hydrogen, non-carbonyl substituentsdirectly bound thereto.

In certain embodiments, the heterobifunctional linker has a molecularweight in the range of 100-10000. For example, the heterobifunctionallinker can have a molecular weight in the range of 100-7000, or100-5000, or 100-2000, or 100-1000, or 100-500, or 200-10000, or200-7000, or 200-5000, or 200-2000, or 200-1000, or 200-500, or500-10000, or 500-7000, or 500-5000, or 500-2000, or 1000-10000, or1000-7000, or 1000-5000.

The term “molecular weight”, as used herein, generally refers to themass or average mass of any of the compositions disclosed herein (e.g.the heterobifunctional linker, the intervening moiety, thechemically-reactive moiety, or any of the entities or moleculescomprising the linkers and moieties described herein). The molecularweight of any of the materials disclosed herein may be calculated as thesum of the atomic weight of each atom in the formula of the materialmultiplied by the number of each atom, and all molecular weightsdisclosed herein are provided as weight average molecular weights. Itmay also be measured by mass spectrometry, NMR, chromatography, lightscattering, viscosity, and/or any other methods known in the art.Relative atomic and molecular mass values are dimensionless, but can begiven the “unit” Dalton (or atomic mass unit) to indicate that thenumber is equal to the mass of one molecule divided by 1/12 of the massof one atom of ¹²C. It is known in the art that the unit of molecularweight may be g/mol, Dalton (Da), or atomic mass unit (amu), wherein 1g/mol=1 Da=1 amu.

In an embodiment, the heterobifunctional linker as disclosed herein hasthe structure:

In a second aspect, the disclosure relates to a composition comprisingthe heterobifunctional linker as disclosed herein, wherein the phosphatemoiety of the heterobifunctional linker is covalently bound to a firstmolecule. In some embodiments, the first molecule comprises a nucleicacid, and the phosphate moiety of the heterobifunctional linker iscovalently bound to a 3′-hydroxyl of a phosphate moiety of the nucleicacid to form a phosphodiester. In some embodiments, the nucleic acidcontemplated comprises about 5 to about 150, or more nucleotides inlength. For example, about 5 to about 90 nucleotides in length, about 5to about 80 nucleotides in length, about 5 to about 70 nucleotides inlength, about 5 to about 60 nucleotides in length, about 5 to about 50nucleotides in length about 5 to about 45 nucleotides in length, about 5to about 40 nucleotides in length, about 5 to about 35 nucleotides inlength, about 5 to about 30 nucleotides in length, about 5 to about 25nucleotides in length, about 5 to about 20 nucleotides in length, about5 to about 15 nucleotides in length, about 5 to about 10 nucleotides inlength, about 400 to about 10,000 nucleotides or more in length, and allnucleic acids intermediate in length of the sizes specifically disclosedto the extent that the nucleic acid is able to achieve the desiredresult. Accordingly, nucleic acids of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or morenucleotides in length are contemplated. In an embodiment, the nucleicacid comprises DNA of a DNA-coated nanoparticle. In certain embodimentsof the second aspect, the chemically-reactive moiety of theheterobifunctional linker is covalently bound to a second molecule.

In a third aspect, the disclosure relates to a composition comprisingthe heterobifunctional as disclosed herein, wherein thechemically-reactive moiety of the heterobifunctional linker iscovalently bound to a second molecule.

In some embodiments of the second or third aspects, the second moleculeis, comprises, or consists essentially of a polypeptide (such as aprotein), a nucleic acid (such as DNA or RNA), a dye, a lipid, a sterol,a fatty acid, and a polymer. For example, the heterobiofunctional linkerdescribed herein, allows one the ability to enzymatically attach anucleic acid molecule to a diverse range of molecules including, but notlimited to, peptides, enzymes, antibody complexes, conjugates, naturalligands, small molecules, quantum dots, radioactive isotopes or chelatesthereof, cytokines, pro-apoptotic substances, pore forming substances,fluorescent proteins (such as green fluorescent protein (GFP, EGFP),blue fluorescent protein (EBFP, EBFP2), cyan fluorescent protein (ECFP,Cerulean, CyPet) and yellow fluorescent protein derivatives (YFP,Citrine, Venus, YPet), monoclonal antibodies, polyclonal antibodies,bifunctional antibodies, dyes, aromatic dyes, fluorophores, fluorescein,rhodamine, cyanine dyes Cy dyes (such as Cy3, Cy5, and the like, or theAlexa™ family of dyes (such as Alexa 488, 500, 514, 532, 546, 555, 568,594, 610, 633, 647, 660, 680, 700, and 750), nucleic acids, DNA, RNA,aptamers, drugs (e.g., cytostatic agents, cytotoxic agents (such as forexample, but not limited to, DNA interactive agents (such as cisplatinor doxorubicin)); taxanes (e.g. taxotere, taxol); topoisomerase IIinhibitors (such as etoposide); topoisomerase I inhibitors (such asirinotecan (or CPT-11), camptostar, or topotecan); tubulin interactingagents (such as paclitaxel, docetaxel or the epothilones); hormonalagents (such as tamoxifen); thymidilate synthase inhibitors (such as5-fluorouracil); anti-metabolites (such as methotrexate); alkylatingagents (such as temozolomide (TEMODAR™), cyclophosphamide); aromatasecombinations; ara-C, adriamycin, cytoxan, gemcitabine, Chlormethine,Ifosfamide, Melphalan, Chlorambucil, Pipobroman, Triethylenemelamine,Triethylenethiophosphoramine, Busulfan, Carmustine, Lomustine,Streptozocin, Dacarbazine, Floxuridine, Cytarabine, 6-Mercaptopurine,6-Thioguanine, Fludarabine phosphate, oxaliplatin, leucovirin,oxaliplatin (ELOXATIN™), Pentostatine, Vinblastine, Vincristine,Vindesine, Bleomycin, Dactinomycin, Daunorubicin, Doxorubicin,Epirubicin, Idarubicin, Mithramycin, Deoxycoformycin, Mitomycin-C,L-Asparaginase, Diethylstilbestrol, Testosterone, Prednisone,Fluoxymesterone, Dromostanolone propionate, Testolactone,Megestrolacetate, Methylprednisolone, Methyltestosterone, Prednisolone,Triamcinolone, Chlorotrianisene, Hydroxyprogesterone, Aminoglutethimide,Estramustine, Medroxyprogesteroneacetate, Leuprolide, Flutamide,Toremifene, goserelin, Cisplatin, Carboplatin, Hydroxyurea, Amsacrine,Procarbazine, Mitotane, Mitoxantrone, Levamisole, Navelbene,Anastrazole, Letrazole, Capecitabine, Reloxafine, Droloxafine, orHexamethylmelamine), prodrugs, radionuclides, imaging agents, polymers,antibiotics, fungicides, anti-viral agents, anti-inflammatory agents,anti-tumor agents, cardiovascular agents, anti-anxiety agents, hormones,growth factors, steroidal agents, microbially derived toxins

Depending on the particular chemistry used, the chemically-reactivemoiety of the linker can react with a moiety that is considered part ofthe second molecule, e.g., a thiol of a peptide, or rather with a moietythat is provided on the second molecule specifically for the purpose ofattachment, e.g., an alkyne or an azide functionalized onto a nucleicacid to provide for an alkyne-azide click reaction.

In certain embodiments of the second aspect,

-   -   (a) the chemically-reactive moiety of the heterobifunctional        linker comprises an azide, and the heterobifunctional linker is        covalently bound to the second molecule through a thiazole        formed from the azide and an alkyne;    -   (b) the chemically-reactive moiety of the heterobifunctional        linker comprises an alkyne, and the heterobifunctional linker is        covalently bound to the second molecule through a thiazole        formed from the alkyne and an azide;    -   (c) the chemically-reactive moiety of the heterobifunctional        linker comprises a cyclopentadiene, and the heterobifunctional        linker is covalently bound to the second molecule through a        cycloaddition product of the cyclopentadiene;    -   (d) the chemically-reactive moiety of the heterobifunctional        linker comprises a thiol, and the heterobifunctional linker is        covalently bound to the second molecule through a thiol-ene or        thiol-yne reaction product of the thiol;    -   (e) the chemically-reactive moiety of the heterobifunctional        linker comprises an alkene (for example, a terminal alkene), and        the heterobifunctional linker is covalently bound to the second        molecule through a thiol-ene reaction product of the alkene;    -   (f) the chemically-reactive moiety of the heterobifunctional        linker comprises a maleimide, and the heterobifunctional linker        is covalently bound to the second molecule through a thiol-ene        reaction product of the maleimide;    -   (g) the chemically-reactive moiety of the heterobifunctional        linker comprises an alkyne (for example, a terminal alkyne), and        the heterobifunctional linker is covalently bound to the second        molecule through a thiol-ene reaction product of the alkyne;    -   (h) the chemically-reactive moiety of the heterobifunctional        linker comprises a carboxylate and the heterobifunctional linker        is covalently bound to the second molecule through a        condensation reaction product of the carboxylate (for example,        with an amine to form an amide);    -   (i) the chemically-reactive moiety of the heterobifunctional        linker comprises an amine and the heterobifunctional linker is        covalently bound to the second molecule through a condensation        reaction product of the amine (for example, with a carboxylate        to form an amide);    -   (j) the chemically-reactive moiety of the heterobifunctional        linker comprises a carboxylate ester of N-hydroxysuccinimide and        the heterobifunctional linker is covalently bound to the second        molecule through a condensation reaction product of the a        carboxylate ester of N-hydroxysuccinimide (for example, with an        amine to form an amide);    -   (k) the chemically-reactive moiety of the heterobifunctional        linker comprises an isocyanate or an isothiocyanate and the        heterobifunctional linker is covalently bound to the second        molecule through a reaction product of the isocyanate or        isothiocyanate (for example, with an amine such as a lysine        amine to form a urea or a thiourea);    -   (l) the chemically-reactive moiety of the heterobifunctional        linker comprises an iodoacetamide and the heterobifunctional        linker is covalently bound to the second molecule through a        reaction product of the iodoacetamide (for example, with a thiol        such as a cysteine thiol to form a thioether); or    -   (m) the chemically-reactive moiety of the heterobifunctional        linker comprises a (pyridin-2-yl)disulfanyl or a        (3-carboxy-4-nitrophenyl)disulfanyl and the heterobifunctional        linker is covalently bound to the second molecule through a        reaction product of the (pyridin-2-yl)disulfanyl or        (3-carboxy-4-nitrophenyl)disulfanyl iodoacetamide (for example,        with a thiol such as a cysteine thiol to form a disulfide        bridge).

In some embodiments of the second or third aspects, thechemically-reactive moiety of the heterobifunctional linker is reactivein a cycloaddition (e.g., a [3+2] cycloaddition or a [4+2]cycloaddition) at a temperature below 60° C., and the heterobifunctionallinker is covalently bound to the second molecule through acycloaddition reaction product of the chemically-reactive moiety.

In a fourth aspect, the disclosure relates to a method (e.g., to form acomposition) of covalently linking two molecules comprising:

(a) reacting the chemically-reactive moiety of the heterobifunctionallinker as disclosed herein with a first molecule comprising a functionalgroup capable of covalently binding the chemically-reactive moiety ofthe heterobifunctional linker, wherein the reacting occurs underconditions and for a time suitable to covalently bind the first compoundto the chemically-reactive moiety of the heterobifunctional linker; and

(b) reacting the phosphate moiety of the heterobifunctional linker witha second molecule comprising a 3′-OH group of a nucleic acid phosphatein the presence of a T4 DNA ligase for a time and under conditions, toligate the second molecule to the phosphate moiety of the first complexto form a phosphodiester bond between the nucleic acid phosphate and thephosphate moiety of the heterobifunctional linker.

In some embodiments of the fourth aspect, step (a) is performed beforestep (b). In an embodiments, the product of step (a) is purified (e.g.,using chromatography) before step (b) is performed. In certainembodiments, step (a) is performed using copper catalysis.

In a fifth aspect, the disclosure relates to a kit comprising:

-   -   (a) the heterobifunctional linker as disclosed herein;    -   (b) reagents for reacting a first molecule to the reactive        moiety of the heterobifunctional linker; and    -   (c) a T4 DNA ligase and reagents to ligate a second molecule to        the phosphate moiety of the heterobifunctional linker.

Optimal amounts of kit reagents to be used in a given reaction can bereadily determined by the skilled artisan having the benefit of thecurrent disclosure. The kits, typically, can be adopted to contain theconstituents aforedescribed in separate packaging or compartments.

When a structure is described as consisting essentially of a particularmoiety, at least 70%, at least 80%, or even at least 90%, of thatstructure by weight is made up of that moiety. The structure canadditionally include, for example, a linker disposed between thatparticular moiety and a reactive group.

EXAMPLES Example 1. Universal Enzyme Responsive Linker for AssemblingLigands on DNA Functionalized Nanomaterials

Enzymatic chemical ligations are a powerful tool by which two DNA ends,one bearing a 3′ hydroxyl group and the other bearing a 5′ monophosphategroup, can be brought together by covalent attachment to form a newphosphodiester bond. DNA ligase is utilized for attaching DNA primersand templates to DNA functionalized substrates for DNA microarray andsequencing applications. To date there is no general enzymatic strategyfor modification of the DNA on a DNA-nanoparticle to impartcompatibility for chemical ligation between the DNA and a molecule ofnon-nucleic acid structure.

Disclosed herein, is a method that utilizes an enzyme-responsiveauxiliary monophosphate cross linker to universally ligate a diversearray of molecules of interest to the DNA on a DNA-nanoparticle scaffold(see FIG. 1).

This approach was developed to address the need for a quick, robust andscalable way to attach biomolecules and other small molecule tags tonanoparticle surfaces without the need for multistep synthesis. Theexamples provided herein demonstrate that this can be achieved underaqueous, physiologically relevant conditions to address many of thesedesired qualities. The ability of the ligase to covalently attach avariety of molecules (i.e. planar, hydrophobic, hydrophilic, charged)shows the enormous potential and versatility of this approach.

A major objective in the design of the heterobifunctional linker was tointerface nucleic acids with a variety of important moieties relevantfor biosensing and therapeutic applications. The four-step synthesisfrom commercially available reagents results in a monophosphorylatedlinker and an azide group that can be used for copper catalyzed “click”reactions (see FIG. 2). It was of interest to sample key moleculesrelevant to diagnostics and therapeutics and therefore a Cy3fluorophore, a cell penetrating peptide, and a second nucleic acid wereall evaluated for their ability to efficiently ligate to the linker.

The approach relies on the ability to engage the active site of theligase and allow space to tether larger molecules such as dyes onto thelinker while achieving sufficient ligation efficiencies. In order toevaluate the extent of ligation on a DNA-nanoparticle surface acombination of dynamic light scattering, agarose gel electrophoresis andfluorescence spectroscopy were utilized. In each instance the moleculeof interest (dye, peptide, or DNA) was first covalently linked to themonophosphate linker using standard copper catalyzed click conditions.Following purification, the molecules were then incubated with aDNA-functionalized nanoparticle in presence of sufficient ATP and T4 DNAligase. Phosphorylated TAT peptide (CKRKKRRKRRRG; SEQ ID NO:06) was ofinterest to generate and ligate to the particle surface as TAT peptideis known to cross the lipid bilayer of a cell due to its high amount ofpositively charged amino acid residues. In order to attach a clickablegroup to the TAT peptide it was synthesized with a terminal cysteineresidue to which a maleimide-alkyne was used to drive a Michael-Additionof the alkyne to the peptide's amino end. (see FIG. 3)

The alkyne modification allowed for covalent attachment of the peptideto the monophosphorylated linker. Once purified, the alkyne modifiedpeptide is attached to the azide of the monophosphorylated linkerforming the tetrazole linkage. The characterization of the particlespost ligation with the linker modified peptide at the DNA-NP surface isshown in FIG. 4.

To test for the ability to attach other molecules an agarose gel shiftassay was utilized to see the relative degree of ligation for variousdifferent chemical groups. FIG. 5 shows the results of a gel shift assaydemonstrating the change in particle size post enzymatic ligation oflinker modified peptide, DNA, and dye. Lane 1 is DNA-gold NPs, lane 2 isDNA-NPs post ligation to DNA strand, lane 3 is post ligation to peptide,and lane 4 is post ligation to Cy3 fluorophore using the universallinker. The ligation reaction conditions were: 24 hours at 37° C. Gelconditions: 1% Agarose, 10 minutes at 200V.

In addition to characterizing the change in the particles surface due tosize and charge changes by dynamic light scattering (DLS) and agarosegel shift respectively, confocal microscopy was also utilized todetermine the effectiveness of the linker conjugation and ability todeliver molecules into cells using this new linkage approach. To date ithas been observed that DNA-functionalized nanoparticles (DNA-NPs) areable to enter cells readily in contrast to their linear nucleic acidcounterparts. When immobilized and when the DNA grafting density issufficient the DNA-NPs can engage scavenger receptors and enter cellswithout transfection agents. In order to test the utility of themonophosphorylated linker, an alkyne modified Cy3 dye was clicked ontothe monophosphate linker and then ligated to the surface of the DNA-NP.This dye conjugated DNA was then incubated with Hela cells and evaluatedfor signs of fluorescence (see FIG. 6).

The results show that the linker is effective at attachingmonophosphorylated material to the surface of the nanoparticle and thatthe nanoparticle formulation is not toxic. Such a versatile approach isimportant for the rapidly growing field of nucleic acid basedtherapeutics and offers an important strategy aimed at repurposingenzymes for use as assembly tools for functionalizing nanomaterials in achemically specific manner.

I claim:
 1. A method of covalently linking two molecules comprising: (a)reacting a chemically-reactive moiety of a heterobifunctional linkerwith a first molecule comprising a functional group capable ofcovalently binding the chemically-reactive moiety of theheterobifunctional linker, wherein the heterobifunctional linker has theformula:(HO)2(O)P—O—I—Y; wherein I is an intervening moiety, the interveningmoiety having a molecular weight in the range of 100 to 10000; and Y isa chemically-reactive moiety, the chemically-reactive moiety beingreactable to couple the linker to an organic compound in aqueoussolution, or a salt thereof, the chemically-reactive moiety andintervening moiety being selected such that the heterobifunctionallinker, or salt thereof, is water soluble at a concentration of at least10 pM at a pH within the range of 6.5 to 7.8; wherein the reactingoccurs under conditions and for a time suitable to covalently bind thefirst compound to the chemically-reactive moiety of theheterobifunctional linker to form a first complex; and (b) reacting thephosphate moiety of the heterobifunctional linker with a second moleculecomprising a 3′-OH group of a nucleic acid phosphate in the presence ofa T4 DNA ligase for a time and under conditions, to ligate the secondmolecule to the phosphate moiety of the first complex to form aphosphodiester bond between the nucleic acid phosphate and the phosphatemoiety of the heterobifunctional linker.
 2. The method of claim 1,wherein step (a) is performed before step (b).
 3. The method of claim 2,wherein the product of step (a) is purified before step (b) isperformed.
 4. The method of claim 1, wherein step (a) is performed usingcopper catalysis.
 5. The method of claim 1, wherein thechemically-reactive moiety is reactable to couple the linker to abiomolecule in aqueous solution.
 6. The method of claim 1, wherein thechemically-reactive moiety comprises an azide.
 7. The method of claim 1,wherein the chemically-reactive moiety comprises an alkyne.
 8. Themethod of claim 7, wherein the alkyne has the structural formula


9. The method of claim 7, wherein the alkyne has the structural formula


10. The method of claim 1, wherein the chemically-reactive moietycomprises a molecule selected from the group consisting of an alkene, anamine, a maleimide, a carboxylate, a carboxylate ester ofN-hydroxysuccinimide, a (3-carboxy-4-nitrophenyl)disulfanyl, acyclopentadiene, an iodoacetamide, an isocyanate, isothiocyanate, a(pyridin-2-yl)disulfanyl, and a thiol.
 11. The method of claim 10,wherein the chemically-reactive moiety comprises an alkene and thealkene is a terminal alkene.
 12. The method of claim 1, wherein thechemically-reactive moiety is reactive in a cycloaddition at atemperature below 60° C.
 13. The method of claim 1, wherein theintervening moiety comprises an entity selected from the groupconsisting of a polyethylene glycol, a polyethyleneimine, a copolymer orcooligomer of ethylene glycol and ethyleneimine, a peptide oligomer orpolypeptide, a polyester of one or more of lactic acid and glycolicacid, a poly(propylene glycol), and a poly(ethylene glycol-co-propyleneglycol).
 14. The method of claim 13, wherein the intervening moietycomprises a polyethyelene glycol having structure —(CH₂CH₂O)_(n)— inwhich n is in the range of 3-200.
 15. The method of claim 13, whereinthe intervening moiety comprises the structure —(CH₂CH₂O)_(n)—(CH₂)_(m)—in which m is in the range of 1-6.
 16. The method of claim 13, whereinthe intervening moiety comprises a polyester of one or more of lacticacid and glycolic acid, and wherein the polyester comprises poly(lacticacid-co-glycolic acid).
 17. The method of claim 1, wherein theintervening moiety comprises the structure —(L)—(CH₂)_(m)— in which m isin the range of 1-6, and in which L comprises a polyethylene glycol, apolyethyleneimine, a copolymer or cooligomer of polyethylene glycol andpolyethyleneimine, a peptide oligomer or polypeptide, a polyester of oneor more of lactic acid and glycolic acid, a poly(propylene glycol), or apoly(ethylene glycol-co-propylene glycol).
 18. The method of claim 1,wherein there are no branched carbon atoms or nitrogen atoms within 4atoms of a branched carbon atom or nitrogen atom being defined as havingmore than three non-hydrogen, non-carbonyl substituents directly boundthereto.
 19. The method of claim 1, wherein the heterobifunctionallinker has a molecular weight in the range of 100-10000.
 20. The methodof claim 1, wherein the heterobifunctional linker has the structure: